More than 140,000 Americans are diagnosed with colorectal cancer (CRC) each year and almost 52,000 individuals die from it. Decreasing the frequency of CRC-related death will undoubtedly require tailoring an individual's treatment to the specific mutations that have occurred in their cancer. Activating mutations in the K-Ras oncoprotein are common in CRC and are associated with particularly poor response to both conventional and targeted therapies. For example, the strong association between mutant K-Ras and resistance to anti-EGFR therapy led the American Society for Clinical Oncology to recommend that all cancer patients undergo K-Ras mutation testing prior to receiving treatment. Nevertheless, new clinical and experimental data have called into question the universality of therapeutic resistance associated with mutant K-Ras. Patients with specific K-Ras activating mutations, such as G13D, appear to benefit from anti-EGFR therapy, just like K-Ras wild-type patients. Building upon our expertise in studying Ras-mutant CRC in mouse models (Haigis et al. Nat. Genet. 40: 600-608, 2008; Wang et al. Cancer Disc. 3: 294-307, 2013), we will perform an in-depth analysis of the relationship between specific K-Ras mutations and therapeutic response. This work is separated into four related specific aims: (1) To comprehensively characterize the K-Ras mutational spectrum and to identify co-occurring mutations in primary human CRCs, (2) To generate an allelic series of Cre-dependent K-Ras activating alleles in mice, (3) To measure the molecular, cellular, and tissue-level phenotypes associated with different K-Ras mutations in a mouse model of CRC, and (4) To evaluate the efficacy of EGFR and MEK inhibitors in mouse models of CRC expressing different mutant alleles of K-Ras. In the end, the K-Ras mutant mouse models developed in this project will reveal genetic subpopulations of CRC patients that will benefit from specific targeted therapies.